Hepatitis C virus afflicts ~2% of the U.S. population, causing acute and chronic liver disease, cirrhosis, liver cancer, and liver failure. Available therapies for treating HCV are poorly tolerated and effective in only a fraction of patients. It is therefore vital that we focus on the development of new therapies to treat HCV infection. The success of this effort hinges on developing an understanding of the molecular mechanisms underlying HCV gene expression and replication, as the enzymes involved these processes are the molecular targets for drug design. The study of these enzymes, particularly in their natural context, has been hampered by the lack of robust in vitro systems to study the assembly and function of the intact replication complex (RC). We lack structural information on the RC and, despite numerous studies on the isolated replicative enzymes (such as the nonstructural proteins NS3 and NS5B) there is growing evidence that these enzymes are highly dependent on cofactors and that they function differently when operating within the RC machine. Therefore, to develop biologically and pharmacologically relevant assays and to learn new information about the HCV replicative enzymes, we propose to isolate the HCV RC and to study its structure and function as an intact replicative holoenzyme. This challenge will be confronted by using two different, complementary approaches: 1. In vitro translation, processing and assembly of the intact RC. 2. Isolation and purification of the RC from liver cell lines. A second limitation to the development of HCV therapeutics is that there is no information on the architecture of the HCV RC. We do not know how the proteins fit together, how the RC integrates into membranes or how it binds the RNA genome. To address these issues, a second goal of our project is to determine sites of intermolecular interaction within the RC and to build a three-dimensional model of the RC complex structure. This effort will provide new insights into the coordinated activities of the RC proteins and it will reveal protein-protein interfaces that can serve as targets for the development of small molecule inhibitors. Three different approaches are being used to build the interaction map for the HCV RC: 1. Biochemical methods combined with high-resolution mass spectrometry. 2. Genetic suppression analysis to identify coupled amino acids. 3. Phylogenetic methods to detect functionally coupled residues from HCV sequence alignments. The success of this effort will be contingent on the coordinated work of chemists, biochemists and viral geneticists that are represented in the project leadership team.

Public Health Relevance

Hepatitis C virus (HCV) is a major cause of acute and chronic liver disease, cirrhosis, liver cancer, and liver failure. An HCV vaccine is not available, and current HCV therapies are inadequate. This proposal addresses the basic mechanisms of HCV genome replication, which are key targets for developing improved antiviral therapies.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01AI089826-05
Application #
8685099
Study Section
Virology - A Study Section (VIRA)
Program Officer
Koshy, Rajen
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
New Haven
State
CT
Country
United States
Zip Code
06510
Kohlway, Andrew; Pirakitikulr, Nathan; Ding, Steve C et al. (2014) The linker region of NS3 plays a critical role in the replication and infectivity of hepatitis C virus. J Virol 88:10970-4
Rawling, David C; Pyle, Anna Marie (2014) Parts, assembly and operation of the RIG-I family of motors. Curr Opin Struct Biol 25:25-33
Kohlway, Andrew; Pirakitikulr, Nathan; Barrera, Francisco N et al. (2014) Hepatitis C virus RNA replication and virus particle assembly require specific dimerization of the NS4A protein transmembrane domain. J Virol 88:628-42
Lindenbach, Brett D; Rice, Charles M (2013) The ins and outs of hepatitis C virus entry and assembly. Nat Rev Microbiol 11:688-700
Appleby, Todd C; Anderson, Robert; Fedorova, Olga et al. (2011) Visualizing ATP-dependent RNA translocation by the NS3 helicase from HCV. J Mol Biol 405:1139-53
Phan, Tung; Kohlway, Andrew; Dimberu, Peniel et al. (2011) The acidic domain of hepatitis C virus NS4A contributes to RNA replication and virus particle assembly. J Virol 85:1193-204
Lindenbach, Brett D (2011) Understanding how hepatitis C virus builds its unctuous home. Cell Host Microbe 9:1-2
Luo, Dahai; Ding, Steve C; Vela, Adriana et al. (2011) Structural insights into RNA recognition by RIG-I. Cell 147:409-22
Lindenbach, Brett D (2010) New cell culture models of hepatitis C virus entry, replication, and virus production. Gastroenterology 139:1090-3